Preparation of visible colored insoluble carrier particles labeled with a fluorescent dye and an immunoassay method using the same

a technology of fluorescent dye and carrier particles, which is applied in the direction of fluorescence/phosphorescence, instruments, material analysis, etc., can solve the problems of large amounts of target substances, ligands or particles cannot be detected by visual observation, and the detection sensitivity cannot be drastically improved, so as to achieve the effect of improving accuracy

Inactive Publication Date: 2019-11-28
DENKA CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0018]The presence of the visible colored insoluble carrier particles labeled with a fluorescent dye of the present invention can be detected by visual observation or with the use of a fluorescence detector. Therefore, when the visible colored insoluble carrier particles labeled with a fluorescent dye of the present invention are used for immunoassay for the measurement of a target substance based on the presence of carrier particles, the presence or absence of the target substance can be determined by visual observation or with the use of a fluorescence detector. The use of the visible colored insoluble carrier particles labeled with a fluorescent dye of the present invention enables the implementation of POCT in a simple manner even in a facility or location in which no equipment such as a fluorescence detector is available. In addition, the use of a fluorescence detector also allows fluorescence measurement with improved accuracy.

Problems solved by technology

However, detection sensitivity would not be drastically improved even by applying these insoluble carrier particles to a dedicated apparatus for detection.
In this case, however, such ligand or particles cannot be detected by visual observation, and large amounts of target substances are necessary.
In addition, even if detection can be carried out within a short period of time upon visual observation, a dedicated apparatus needs to be used, which disadvantageously results in complicated detection procedures.
Therefore, if the amount of a target substance is very small, ligands on fluorescent particles cannot bind to the target substance, and this does not always result in high sensitivity.
This is problematic due to poor efficiency.
In this case, special techniques are required to prepare such particles, which is also problematic.

Method used

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  • Preparation of visible colored insoluble carrier particles labeled with a fluorescent dye and an immunoassay method using the same
  • Preparation of visible colored insoluble carrier particles labeled with a fluorescent dye and an immunoassay method using the same
  • Preparation of visible colored insoluble carrier particles labeled with a fluorescent dye and an immunoassay method using the same

Examples

Experimental program
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Effect test

example 1

on of Visible Colored Fluorescent Particles Labeled with a Fluorescent Dye with the Use of Colored Polystyrene Particles Having a Carboxyl Group as a Functional Group

1. Binding of a Europium Chelate Fluorescent Labeling Agent

[0047]Different color types of visible colored polystyrene particles having a carboxyl group as a functional group (see Table 1 and FIGS. 1-3) were diluted with a 10 mM HEPES (pH 7.0) buffer solution to obtain a concentration of 0.1%. ATBTA-Eu3+ (Tokyo Chemical Industry Co., Ltd.) serving as a europium chelate fluorescent labeling agent was added thereto to obtain a concentration of 0.1 mg / mL, followed by stirring. Thereafter, carbodiimide was added thereto to obtain a concentration of 1%, followed by stirring. The resultant was allowed to react at room temperature for 1 hour, followed by centrifugation at 7000 g for 30 minutes. Thereafter, the supernatant was removed and the resultant was resuspended in a solution containing 50 mM Tris (pH 9.0) and 3% BSA.

2. Bi...

example 2

f an Anti-Influenza a Virus NP Antibody and a Fluorescent Labeling Agent to Visible Colored Polystyrene Particles Having a Carboxyl Group as a Functional Group and Detection of an Influenza a Virus NP Antigen Through Immunochromatography Based on Such Binding

[0055]In Example 2, visible colored polystyrene particles were compared. Verification was carried out using, as materials, red polystyrene particles and blue polystyrene particles I, both of which showed high visibility.

1. Preparation of Anti-Influenza a Virus NP Monoclonal Antibody

[0056]A BALB / c mouse was immunized with an influenza A virus antigen and raised for a premeasured time. The spleen was excised out from the mouse and the spleen cells were fused with mouse myeloma cells (P3×63) in accordance with the Kohler's method (Kohler et al., Nature, vol. 256, p 495-497 (1975)). The resultant hybridoma cells were kept in an incubator of 37° C. While the antibody activity of the supernatant was checked by ELISA using a plate on w...

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Abstract

The object of the present invention is to provide insoluble carrier particles used for high-sensitivity rapid immunoassay, which allow visual observation and high-sensitivity apparatus measurement.The insoluble carrier particles are visible colored insoluble carrier particles labeled with a fluorescent dye, which are used for immunoassay, wherein absorption of fluorescence by the visible colored insoluble carrier particles is low within a wavelength range of fluorescence emitted by the fluorescent dye.

Description

TECHNICAL FIELD[0001]The present invention relates to a method for detecting an analyte contained in a sample in a specific and rapid manner and a kit using the method.BACKGROUND ART[0002]Hitherto, immunoassay methods using insoluble carrier particles have been widely performed.[0003]Immunoassay methods using insoluble carrier particles comprise the following steps: preparing a test piece on which a ligand capable of specifically binding to a target substance has been immobilized at a certain site on a membrane having a fiber structure such as a nitrocellulose membrane; allowing insoluble carrier particles (e.g., gold colloid, platinum-gold colloid, and / or colored polystyrene particles) on which a ligand capable of specifically binding to a target substance has been immobilized and a sample containing a target substance to be developed on the test piece with the use of a capillary phenomenon, thereby causing the target substance to be sandwiched between the ligand on the nitrocellul...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): G01N33/58G01N33/533G01N21/64
CPCG01N2021/6439G01N33/533G01N21/6428G01N33/582
Inventor MIYAZAWA, TAKASHI
Owner DENKA CO LTD
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